Physiological control of the concentration of free fatty acid levels is central to human physiology. Fatty acid binding proteins (FABPs), a large superfamily of proteins found abundantly in many cell types, have been shown to play a role in the organism's ability to control fatty acid levels by selectively binding fatty acids. The details of the ability of these proteins to select fatty acids, based on chain length, saturation state, and headgroup type, is not clear at a molecular level. This is despite the existence of two central ingredients required for such an explanation: highly detailed structures (Banaszak and Cistola) and excellent binding thermodynamics (Kleinfeld). We aim, through this grant proposal, to provide the third needed ingredient for a detailed molecular description of binding. That is, our overall goal is the ability to compute the selective binding of fatty acids within fatty acid binding proteins. We approach this goal through three specific aims. Our first aim is to understand the basis of fatty acid solvation in the homogenous solvents water and octanol. This will provide a reference point for the changes to a heterogeneous environment provided by the fatty acid binding protein and considered in the next two aims. In the second aim, we will directly compute the relative free energy differences underlying discrimination of fatty acids afforded by the intestinal fatty acid binding protein (I-FABP). In the third aim we will draw connections between our calculations and the site directed mutants that have been characterized in the Kleinfeld lab. Throughout these aims we will use alchemical mutagenesis calculations with a range of solvation models. In particular, we will be testing new models for solvation developed in the Roux lab. The work will further involve extending our own approaches to relative free energy calculations through dynamic importance sampling, analysis of non-equilibrium work events, extrapolations from histogram analysis and intelligent stepping algorithms along the alchemical reaction coordinate. If we succeed in our long term goal, we will have achieved the ability to rationally design new protein binding interactions enabling discrimination of particular fatty acids. The achievement of this goal would have implications for rational treatment of heart disease and other, e.g. digestive system related, human diseases that involve the control of free fatty acid levels.